Tamoxifen is the major SERM (Selective Estrogen Receptor Modulator) used in breast cancer. However, patient response to tamoxifen is variable and tamoxifen exhibits serious estrogen-like side effects in nontarget tissues such as uterus. At present, there is no clear mechanistic explanation for this mixed activity of tamoxifen in different tissues. The estrogen receptor (ER) interacts with a diverse class of proteins termed coregulators, both coactivators and corepressors that, respectively, enhance or inhibit ER action. Recent reports strongly suggest that hormonal signaling pathways promote the estrogen agonist action of tamoxifen through phosphorylation of ER and coregulators. This proposal hypothesizes that the estrogen-like action of tamoxifen in uterine endometrium is regulated, in part, by specific patterns of estrogen receptor and nuclear receptor coregulator phosphorylation that are permissive for functional, estrogen-like interactions between tamoxifen-ER, coactivators and ER target genes. The following Specific Aims will be addressed. Aim 1 will identify how activation and inhibition of the Src and PKA signaling pathways regulates tamoxifen effects on ERalpha-dependent gene expression and identify the specific downstream kinases involved. Aim 2 will identify the phosphopeptide fingerprint of ERalpha and coregulators that is associated with tamoxifen agonist or antagonist action in breast and endometrium-derived cell lines and determine how the Src and PKA pathways alter this phosphorylation. Aim 3 will determine how specific phosphorylation sites in ERalpha and coregulators that are associated with tamoxifen agonist vs. antagonist action contribute to tamoxifen-regulated gene expression in breast and endometrium-derived cell lines. Aim 4 will mechanistically address what affect the Src and PKA pathways and ER and coregulator phosphorylation have on interaction between ER and coregulators, interaction of ER and coregulators with ER-dependent promoters, and coregulator transcriptional coactivation and repression. These studies seek to identify phosphorylation fingerprints of ER and coregulators that would link specific phosphorylation sites to tamoxifen agonist or antagonist action. Elucidation of the mechanisms that govern the mixed activity of tamoxifen is critical for implementation of better tamoxifen therapy regimens for breast cancer and will provide a strong experimental basis for the rational design of novel SERMs with a desired clinical profile.